An Approach to the Genetics of NUE in Maize

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DOI: 10.1093/jxb/erh006

FOCUS PAPERFOCUS PAPER

An approach to the genetics of nitrogen use ef®ciency in

maize

A. Gallais1,* and B. Hirel2

1 Station de Ge Â ne Â tique Ve Â ge Â tale, INRA-UPS-INAPG, Ferme du Moulon, 91190 Gif/Yvette, France 2 Unite Â de Nutrition Azote Â e des Plantes, INRA route de St Cyr, 78026 Versailles Cedex, France

Received 2 April 2003; Accepted 24 July 2003

Abstract

To study the genetic variability and the genetic

basis of nitrogen (N) use ef®ciency in maize, a setof recombinant inbred lines crossed with a tester

was studied at low input (N±) and high input (N+)

for grain yield and its components, grain protein

content, and post-anthesis nitrogen uptake and

remobilization. Other physiological traits, such as

nitrate content, nitrate reductase, glutamine synthe-

tase (GS), and glutamate dehydrogenase activities

were studied at the level of the lines.

GenotypeQnitrogen (GQN) interaction was signi®-

cant for yield and explained by variation in kernel

number. In N±, N-uptake, the nitrogen nutrition

index, and GS activity in the vegetative stage were

positively correlated with grain yield, whereas leaf

senescence was negatively correlated. Whatever N-

input, post-anthesis N-uptake was highly negatively

related to N-remobilization. As a whole, genetic vari-

ability was expressed differently in N+ and N±. This

was con®rmed by the detection of QTLs. More QTLs

were detected in N+ than in N± for traits of vegeta-

tive development, N-uptake, and grain yield and its

components, whereas it was the reverse for grain

protein content and N-utilization ef®ciency. Several

coincidences between genes encoding for enzymes

of N metabolism and QTLs for the traits studied

were observed. In particular, coincidences in threechromosome regions of QTLs for yield and N-remo-

bilization, QTLs for GS activity and a gene encoding

cytosolic GS were observed. This may have a

physiological meaning. The GS locus on chromo-

some 5 appears to be a good candidate gene which

can, at least partially, explain the variation in nitro-

gen use ef®ciency.

Key words: Glutamate dehydrogenase, glutamine

synthetase, maize, nitrate content, nitrate reductase,

nitrogen uptake, nitrogen use ef®ciency, remobilization.

Introduction

The absorption of nitrogen by plants plays an important

role in their growth. Consequently, nitrogen fertilization

has been a powerful tool for increasing the yield of

cultivated plants, such as cereals. Nowadays, both to avoid

pollution by nitrates and to maintain a suf®cient pro®t

margin, farmers have to reduce the use of nitrogen

fertilizer. These objectives can be met through ef®cient

farming techniques, but also by using plant varieties that

have a better nitrogen use ef®ciency (NUE). The devel-opment of such varieties will be more ef®cient with a

better knowledge of the physiological and genetic bases of

NUE.

For grain maize NUE has been de®ned as the grain yield

per unit of nitrogen available from the soil, including

nitrogen fertilizer (Moll et al., 1987). It is the product of

nitrogen uptake ef®ciency (N-uptake/N from soil), and

nitrogen utilization ef®ciency (NUtE, i.e. yield/N-uptake).

For NUE, genetic variability and genotypeQnitrogen

fertilization level interactions re¯ecting differences in

responsiveness have been observed in several studies on

maize (Beauchamp et al., 1976; Pollmer et al., 1979; Balko

and Russell, 1980a; Reed et al., 1980; Russell, 1984; Mollet al., 1987; Landbeck, 1995; Bertin and Gallais, 2000). In

addition, it has been found that correlations among various

agronomic traits such as grain protein yield and its

components are very different according to the level of

nitrogen fertilization (Balko and Russell, 1980b; Di Fonzo

et al., 1982; Rizzi et al., 1993; Bertin and Gallais, 2000).

At high N-input, genetic variation in NUE was explained

* To whom correspondence should be addressed. Fax: +33 1 6933 2340. E-mail: [email protected]

Journal of Experimental Botany , Vol. 55, No. 396, ãSociety for Experimental Biology 2004; all rights reserved

Journal of Experimental Botany, Vol. 55, No. 396, pp. 295±306, February 2004



by variation in N-uptake, whereas at low N-input, NUE

variability was mainly due to differences in nitrogen

utilization ef®ciency. This suggests that the limiting steps

in N-assimilation may be different when plants are grown

under high or low levels of nitrogen fertilization.

Differences in N-uptake are likely to be related to the

quantity and the quality of the root system. However, while

experiments have shown a variability in the architecture of the root system (HeÂbert et al., 1992), it has not been related

to variability in N-uptake. It remains therefore to ®nd

which traits control N-uptake. Nitrogen uptake at silking

determines kernel number (Di Fonzo et al., 1982; Muruli

and Paulsen, 1981; Sherrard et al., 1986). This can be

explained by the high demand for nitrogen of embryos just

after fertilization (Czyzewicz and Below, 1994). As a

consequence, kernel number is very susceptible to a N-

stress in comparison to kernel weight (Uhart and Andrade,

1995; Reed et al., 1988; Below, 1995). Furthermore, Di

Fonzo et al. (1982) and Moll et al. (1987) have shown that

the role of post-anthesis N-uptake in grain ®lling can be

related to leaf senescence. Indeed, by increasing leaf longevity, thus prolonging the capacity of the plant to

absorb mineral nitrogen, better yields were obtained in

modern hybrids (Tollenaar, 1991; Ma and Dwyer, 1998;

Racjan and Tollenaar, 1999a, b).

Although the agronomic studies on maize have demon-

strated that there is genetic variability for NUE, present

knowledge on the corresponding physiological traits is still

limited. Several studies have attempted to assign a role for

the different proteins and enzymes involved in mineral N-

uptake, assimilation and recycling (Lea and Ireland, 1999).

However, most of these approaches involving either whole

plant physiology or the use of transgenic plants or mutants

have not contributed to an understanding of the physio-

logical and genetic basis of NUE in a more integrated

manner.

Nowadays, quantitative genetic studies associated with

the use of molecular markers may be a way of identifying

Quantitative Trait Loci (QTL) involved in the genetic

variation of a complex character such as NUE.

Coincidences between QTLs for agronomic traits and

QTLs for physiological traits related to NUE will give a

physiological meaning to the QTLs for the agronomic

traits. In addition, if there is co-mapping with genes

encoding enzymes involved in N-assimilation, this will

give a genetic meaning to these QTLs, thus allowing theidenti®cation of so called `candidate' genes, i.e. genes for

which allelic variation could be responsible for a part of

the observed variation. It is also possible to identify new

genes as potential candidates by the studies of gene

expression. Having identi®ed a good candidate gene, to

validate it, the favourable allele can be transferred to a

genotype with an unfavourable allele to test whether there

is the expected effect. Although QTLs for adaptation to

environmental stresses such as drought resistance (Agrama

and Moussa, 1996; Ribaut et al., 1997; Tuberosa et al.,

1998), and tolerance to phosphorus stress (Reiter et al.,

1991) have already been detected in maize, few studies

have been published on the identi®cation of QTLs for

adaptation to low N-input. Agrama et al. (1999) found

common and speci®c QTLs for high and low N-input

whereas Bertin and Gallais (2001) clearly showed that

QTLs detected at high N-input were different from thosedetected at low N-input. With the same material as Bertin

and Gallais, coincidences between QTLs for agronomic

traits and QTLs for some physiological traits related to

nitrogen assimilation were studied by Hirel et al. (2001).

The observed coincidences of QTLs for yield and kernel

weight with QTLs for glutamine synthetase (GS) activity

led to the proposal that GS plays an important role in the

determination of yield.

In this paper, some of the results of Bertin and Gallais

(2000, 2001) and Hirel et al. (2001) are reviewed and

discussed, with emphasis being given to traits related to

NUE and to coincidences between QTLs for agronomic

and QTLs for some physiological traits as well as to theircoincidence with genes involved in nitrogen metabolism.

In addition, new results are given for N-remobilization and

post-anthesis N-uptake.

Materials and methods

Plant material and experiments

For QTL detection it is necessary to use a material where correlationamong non-homologous genes can only be due to physical linkage.For the ®eld studies, a random set of 99 recombinant inbred lines(RIL) has been used from 145 that were derived from the crossbetween a French ¯int and early line (F2) and an iodent late line. For

the agronomic study, they were crossed to an unrelated tester (F252)in order to study NUE at the hybrid level, the chosen testercombining well with both parents. Such a population was chosenbecause its two parents are highly complementary in terms of grainproductivity, i.e. heterotic. Furthermore, the agronomic study of theparents revealed differences in their NUE. As described by Bertin(1997) and Bertin and Gallais (2000), two N-levels were used: anormal nitrogen level (N+) with 175 kg N ha±1 applied at the time of sowing and no nitrogen fertilization (N±), all the N being supplied bythe soil, a supply estimated to be at least 50±60 kg ha±1. Theobjective was to reduce the yield by about 40%. The experiment wasdeveloped in two consecutive years, 1994 and 1995, in the samelocation (`Le Moulon' Plant Breeding Station) with two-row plots of 5.2 m in length and 0.80 m between rows and an average of 95 000plants ha±1. Several traits were measured at ¯owering and grainharvest. In the present study, traits used for both correlation studiesand QTL detection were grain yield and its components (kernelnumber plant±1 and kernel weight, i.e. thousand kernel weight), grainnitrogen content and grain nitrogen yield. For more details about theprocedures used to measure these agronomic traits, see Bertin andGallais (2000). Furthermore, traits related to NUE were consideredin particular. At ¯owering they are nitrogen uptake, nitrogen content,and nitrogen nutrition index (NNI, see below), and nitrogen uptakein the whole plant (aerial part). At grain harvest they are nitrogenuptake allocated to the grain and the stover, grain and stover nitrogencontent, nitrogen harvest index, and apparent N-remobilization fromthe aerial biomass, the leaf blade and the stem to the grain derived

296 Gallais and Hirel



from N-quantity in the corresponding organ at ¯owering minus N-quantity at maturity. NNI is de®ned as the ratio of observedN-content to a critical N-content corresponding to the minimum N-content allowing the maximum growth (Lemaire and Gastal, 1997).This index allows a correction for the dilution effect.

Due to the cost of the studies, physiological studies weredeveloped (Hirel et al., 2001) on only 77 RILs randomly chosenfrom the 99 lines used to perform the agronomic studies. Plants weregrown in hydroponic culture with a nutrient solution containing1 mM NO3

± which corresponds to a high N-input. The plants wereharvested at the 6±7 leaf stage and separated into shoots, stems androots. The experiment was replicated over two consecutive years(1998 and 1999). Leaf nitrate content, leaf NADH-nitrate reductase(NR) activity, and leaf GS activity were selected as representativemarker metabolites and enzyme activities of primary N-assimilationin young developing plants. Furthermore, in 2000, from the set of 99RILs evaluated in the ®eld at two nitrogen levels, GS and glutamatedehydrogenase (GDH) activities were studied in the leaf below theear, which corresponds to one of the main origins of carbon andnitrogen assimilates exported to the grain in adult plants (Prioul andSchwebel-DugueÂ, 1992).

Gene mapping

For the mapping of QTLs, the RFLP genetic map published byCausse et al. (1996) containing 152 marker loci corresponding to atotal map length of 1813 cM was used. The mean interval betweentwo markers varies from 8 cM to 18 cM. To identify some candidategenes, speci®c genes involved in N-metabolism were also mapped: ahigh af®nity nitrate transporter, NTR1 (Trueman et al., 1996); twoNADH-NR, NR1 and NR2 (Long et al., 1992); nitrite reductase, NiR

(Lahners et al., 1988); glutamate dehydrogenase, GDH1 (Sakakibaraet al., 1995); four cytosolic GS, gln1, gln2, gln3, and gln4, plastidicGS, gln5 (Sakakibara et al., 1992a); and asparagine synthetase ( AS1

and AS2) (Chevalier et al., 1996) which was located on two loci. Theloci corresponding to gln1, 2, 3, 4, and 5 correspond to the GS genesnamed pGS122, pGS134, pGS107, pGS112, and pGS202 bySakakibara et al. (1992a) and GS1-1, GS1-2, GS1-4, GS1-3, andGS2 by Li et al. (1993).

QTLs were detected using the PlabQTL software (Utz and

Melchinger, 1995) following simple interval mapping. Only QTLswith a LOD score greater than 2 were considered. To take intoaccount the error in location, location of a QTL on the map isrepresented by the chromosome region corresponding to themaximum LOD minus 1, which determines an approximatecon®dence interval (Lander and Botstein, 1989). Two QTLs of different traits will be declared as coincident when their LOD-1

intervals largely overlap. A coincidence will be said to be positivewhen there is coincidence of a favourable (or an unfavourable) allelefor both traits. The coincidence will be said to be negative whenthere is coincidence of a favourable allele for one trait with anunfavourable allele for the other trait.

Statistical analysis

To understand how the plant functions at high and low N-input,phenotypic correlations (r P), i.e. correlations among genotypicmeans, between agronomic traits in each situation and betweenagronomic traits and physiological traits were calculated. Thephenotypic correlations given in this paper were highly signi®cant(greater than 0.26 or 0.29, the thresholds at 0.01, and noted with twoasterisks **). It must be underlined that, due to environmental errors,the correlation can be much lower than genotypic correlation whichwould be calculated on true unknown genotypic values. When themeasurements are independent, as with an agronomic and aphysiological trait, then it is only necessary to divide r P by thesquare root of the product of the heritabilities (Becker, 1984). Such

genotypic correlations (r G) have been derived for some pairs of traitsand were given in detail by Bertin and Gallais (2000). Broad-senseheritabilities were derived from the ratio of genotypic variance tophenotypic variance among lines estimated from the analysis of variance, considering genotypic effects as random.

Results and discussion

Understanding genetics and physiology of N-utilization from the study of N-stress on trait means and correlations between traits

Effect of nitrogen deprivation on trait means: The effect of

nitrogen deprivation on traits related to growth and

development gives preliminary information for under-

standing plant reaction to nitrogen fertilization. In the

experimental conditions used by the authors the reduction

in yield from high to low N-input was 38%. Among the

yield components, kernel number was the most affected

(32%) while kernel weight was reduced by only 9%. The

reduction in kernel number is due to ovule abortion after

fertilization, since the number of ovules is only slightly

affected by nitrogen stress (Lemcoff and Loomis, 1986;

Uhart and Andrade, 1995; Below, 1995; Below et al.,

2000). Such abortion could be the result of a limitation in

the source of photosynthetic products, which also affects

post-anthesis growth (29% reduction) much more than

vegetative development (14% reduction), as already

shown by McCullough et al. (1994). This suggests that,

just after fertilization, the sink demand must be too high

compared with the availability of resources, thus leading to

embryo abortion in a genotype-dependent manner.

Genetic variation in NUE in relation to fertilization: At a

given level of nitrogen, differences in yield means that

there are differences in NUE among different genotypes.

Genetic variance for NUE has been observed both at low

and high nitrogen fertilization levels. The genetic correl-

ation between the two levels was high for both years

(0.85). On average, the variance of genotypeQnitrogen

interaction represented about 25% of the total genotypic

variation. Surprisingly, this result is comparable to that

already observed by various authors on more diverse plant

material (Balko and Russell, 1980a; Landbeck, 1995).

Kernel number per ear was the yield component explaining

the most about such an interaction, underlining once again

the role of embryo abortion just after ovule fertilization.Responsiveness of yield and kernel number was negatively

related to traits at low N-input: yield or kernel number, N-

content at ¯owering, nitrogen nutrition index (NNI), N-

uptake at harvest, and post-anthesis N-uptake. This

observation means that genotypes exhibiting low agro-

nomic performance at low N-input, i.e. those having a low

NUE, were those reacting more to nitrogen fertilization.

Thus, genotypeQnitrogen interaction appears to be essen-

tially due to variation in the adaptation of the plant to low

Nitrogen use ef®ciency in maize 297



N-input rather than to variation in the adaptation to high N-

input. The absence of such interactions for traits relative to

vegetative growth means that they are due to grain

development.

N-uptake is the product biomassQN-content. Its vari-

ation depends on the correlation between the two

components. At ¯owering, under low N-input, there was

a strong negative correlation between yield and N-content(dilution effect), resulting in a strong reduction of genetic

variance of N-uptake. Until ¯owering, maize functions like

a typical forage grass for which a negative correlation

between dry-matter yield and protein content is generally

observed (Lemaire and Gastal, 1997). At maturity, the

observed low variation in whole plant N-uptake under low

N-input suggests that there was a limiting factor in

nitrogen availability in the soil and in the capacity of the

plant to absorb nitrogen. Unlike low N-input, at high N-

input there was a strong variation in N-uptake, with no

negative correlation between yield and N-content.

Since only the aerial biomass is usually taken into

consideration in the de®nition of NUE, nitrogen utilizationef®ciency (NUtE) can be expressed as the ratio of harvest

index/N-content of the aerial parts. In agreement with such

an expression, these results showed that NUtE was highly

negatively related to the plant N-content and positively

related to the harvest index. An increase in NUtE

corresponds with a decrease in the N-content and with an

increase in the harvest index. These results were similar to

that obtained by Di Fonzo et al. (1982) who showed that

under a low level of N fertilization, grain yield was related

to the nitrogen harvest index.

Relationships between nitrogen remobilization and post-

¯owering absorption: It appeared that, regardless of the

level of N fertilization, there was strong opposition

between N-remobilization and post-anthesis N absorption

(r P= ±0.80**). Remobilization from the stem appeared to

be moderately correlated to remobilization from leaf

blades (r P=0.46**) and, as expected, the amount of

nitrogen remobilized was always related to the quantity

of nitrogen present at ¯owering. Whatever the organ, the

relative remobilization (or rate of remobilization) was

highly related to the absolute remobilization (r P=0.88**

under high N input). Grain yield and protein grain yield

were positively correlated with absorption at high N-input,

con®rming again the major role of N-uptake under thesegrowth conditions whereas, as a consequence of the strong

negative correlation between remobilization and absorp-

tion, they were negatively correlated to remobilization.

The opposition between N-remobilization and N-

absorption can be explained by the fact that N-remobiliza-

tion comes essentially from leaf protein degradation

(Rubisco in particular) in senescing leaves while photo-

synthetically ef®cient leaves (with an active Rubisco) are

required for an ef®cient N absorption. Therefore, it can be

assumed that nitrogen remobilization takes place when

absorption is reduced or stopped following various biotic

or abiotic stresses, including water stress, or during natural

senescence.

Relationships between grain yield, NUE and some speci®c

traits. (1) Relationships with vegetative development and

NNI at ¯owering: Whatever the N-input, high vegetativedevelopment at ¯owering was favourable to high nitrogen

uptake ef®ciency. As post-anthesis N-uptake explains a

great part of the genetic variability of the total absorption,

it can be assumed that high vegetative development,

including roots, is necessary to have high nitrogen

absorption during grain ®lling. Such vegetative develop-

ment allows remobilization to be greater if soil-N avail-

ability is restricted. At both low and high N-input, grain

yield was signi®cantly correlated with N-uptake at

¯owering, and more intensively at low N-input where it

determines kernel number. In this condition, it was also

related to NNI at ¯owering (r P=0.45**). Interestingly, at

low N-input, NNI at ¯owering was related to leaf senescence although this process occurred about 3±4

weeks later (r G=0.80). These observations led to the

proposal that NNI re¯ects the physiological status of the

leaf, such as its potential photosynthetic activity. In other

words, with active chloroplasts, leaf N-content is higher

and thus delays leaf senescence. Consequently, a low NNI

limiting the ¯ux of photosynthesis products at ¯owering

can lead to embryo abortion just after fertilization. Such a

conclusion is also supported by the results obtained by

Uhart and Andrade (1995), Reed et al. (1988) and

Czyzewicz and Below (1994), who showed that nitrogen

is necessary for kernel development, perhaps through the

supply of carbon assimilates. Genetic variation in respon-

siveness of kernel number could mean a resistance to

abortion, due for example to the ability to remobilize stalk

reserves.


traits. (2) Relationships with anthesis±silking interval:

Anthesis±silking interval (ASI) is de®ned as the difference

between silking date and anthesis date. From a physio-

logical point of view, the observed negative relationship

between grain yield and ASI (rG= ±0.81), also observed in

other experiments (A Gallais, B Hirel, unpublished results)

and by La®tte and Edmeades (1995), is interesting to note.When maize plants are subjected to various stresses such

as drought or nitrogen de®ciency, there results an increase

in ASI (La®tte and Edmeades, 1995). The consequence is

that in monogenotypic stands there could be a de®cit in

ovule fertilization. In the authors' experiment, this

expected effect was suppressed by a continuous pollen

production during silking except for late genotypes. ASI

could then have a physiological meaning in relation to

stress tolerance. In other words, genotypes for which ASI




does not increase would have a more ef®cient nitrogen

metabolism, or a physiology leading to greater yield at low

N-input. It is well known that a short ASI is related to a

proli®c physiology. At the extreme, true proli®cacy leads

to protogyny whereas normal maize shows protandry.

Such genotypes have two favourable traits. First, they have

a high degree of translocation from the stover to the grain,

a characteristic which favours yield in stress conditions;this explains why, with such material, NUtE is more

important than N-uptake, unlike normal maize (Jackson

et al., 1986). Second, Bertin et al. (1976) and Boyat and

Robin (1977) have shown that they have a higher NR

activity, which is already induced in the leaf at low light

intensity.


traits. (3) Relationships with nitrate absorption in young

vegetative plants: In many plant species, when nitrate is

absorbed in excess it is usually stored in the vacuole and

serves both as an osmoticum and as a source of mineral

nitrogen when the soil supply becomes depleted

(McIntyre, 1997; Crawford and Glass, 1998). During

vegetative growth in maize, the vacuolar pool of nitrate

constitutes an important source of nitrogen that can be

metabolized further and subsequently participates in the

grain-®lling process (Teyker et al., 1989; PleÂnet and

Lemaire, 1999). In this experiment, leaf nitrate content and

leaf nitrate quantity in young developing plants was related

to grain and protein yield, mainly through a strong

relationship with kernel weight (Hirel et al., 2001). The

correlation between kernel weight and nitrate uptake in

young vegetative plants could be due to a greater vigour of

the plants from a heavy kernel, a vigorous plant absorbingmore nitrates than a non-vigorous one. However, in the

®eld, the effect of seed size on plant development tends to

disappear before the 4±5 leaf stage. Therefore, the nitrate

content in young developing plants could be considered as

a good `metabolic marker' of nitrate uptake ability in ®eld-

grown plants. This hypothesis was con®rmed by the

positive correlation between leaf nitrate content (and

quantity) in young vegetative plants and post-anthesis

nitrogen uptake of adult plants under low N-input

(r P=0.29**). Under this condition, leaf nitrate content

was negatively related with nitrogen remobilization. This

can be interpreted as a consequence of the observed

negative correlation between N-remobilization and post-

anthesis N-uptake. However, when soil nitrogen availabil-

ity becomes limiting, such a negative correlation could be

the result of a high N-uptake before ¯owering by ef®cient

absorbing genotypes. At high N-input no correlation was

observed between leaf nitrate content or quantity and

remobilization or post-anthesis N-uptake. This could mean

that, as nitrogen from soil is not limiting, it can be absorbed

at any time as long as the plant does not senesce.


traits. (4) Relationships with GS at young stage and GDH

at mature stage: GS (EC 6.3.1.2) is one of the main

enzymes involved in the assimilation and recycling of

mineral nitrogen which catalyses the ATP-dependent

conversion of glutamine into glutamate utilizing ammonia

as a substrate (Lea and Ireland, 1999; Cren and Hirel,

1999). The authors' working hypothesis was that the rateof ammonium assimilation derived from nitrate reduction

and/or organic nitrogen recycling is of major importance

for plant NUE. The role of GS1 during N-remobilization

has already been shown in maize hybrids containing lower

amounts of nitrate, suggesting that the active contribution

of cytosolic GS during the recycling of nitrogen results

from protein hydrolysis (Purcino et al., 1998). In these

studies (Hirel et al., 2001), leaf GS activity was positively

correlated with grain yield and kernel number under low

N-input and to GNY (r P=0.28**) at high N-input. As N-

remobilization has been shown to play a greater role at low

N-input, this could appear to be consistent with this study's

assumption. However, N-remobilization throughout thegrowth period after ¯owering can affect grain ®lling, i.e.

kernel weight, but not kernel number, which is determined

very early just after fertilization, unless a high GS activity

allows the synthesis of a high level of glutamine derived

compounds just after ¯owering. In fact, leaf GS activity in

young vegetative plants was positively correlated both to

the post-anthesis N-uptake and to the percentage of

nitrogen in the grain from post-anthesis N-uptake (and

then negatively correlated with the percentage of nitrogen

in the grain coming from remobilization) at high N-input.

This observation is also consistent with the correlation

between GS activity and nitrate content or quantity in

young developing plants. The relationship with the kernel

number is likely to be due to a greater ¯ux of nitrates and of

nitrogen compounds in genotypes absorbing higher

amounts of nitrate. The whole leaf GS activity (plasti-

dic+cytosolic) measured in young vegetative plants at high

N-input then appears to be related to N-uptake ability

rather than to N-remobilization. Ammonium recycling

during remobilization could be catalysed by another

cytosolic GS isoenzyme which becomes the predominant

form of the enzyme after ¯owering (B Hirel, unpublished

data).

If GDH (E.C.1.4.1.2) activity is considered, which is

another enzyme which is able to aminate 2-oxoglutarate ordeaminate glutamate to release ammonium and is induced

in senescing leaves (Dubois et al., 2003), the results are

more dif®cult to interpret. It is mainly because the exact

function of the enzyme in vivo is not fully de®ned. Leaf

aminating GDH activity of adult plants measured in vitro

was positively related to kernel number at low N-input

(rP=0.27**). It can therefore be hypothesized that, under

these conditions, the enzyme, in conjunction with GS, may

participate in the reassimilation of ammonium released




following protein hydrolysis during the process of N-

remobilization. Aminating GDH activity could be inter-

preted as an adaptation to a shortage of nitrogen.

Deaminating GDH activity at low N-input was negatively

correlated with GS activity, with the amount of nitrogen

accumulated at anthesis, and with the leaf N-content at

maturity. Dubois et al. (2003) suggest that its function

would be more for carbohydrate replenishment rather thanN-assimilation. However, one cannot completely exclude

the dual function of the enzyme: depending on the nitrogen

status of the plant it could act as a signal to control the

homeostasis of glutamate (the substrate of GS) and thus the

¯ux of reduced N.


traits. (5) Nitrate reductase activity: As expected, a

negative correlation was observed between NR activity

and nitrate content in young plants (r P= ±0.32**).

Whatever N-input, NR activity was related negatively to

kernel weight and N reduction ef®ciency was negatively

related to both grain yield and grain protein yield. In otherwords, high NR activity and nitrogen reduction ef®ciency

characterize the less yielding genotypes (Hirel et al.,

2001). Similar conclusions were drawn by Reed et al.

(1980) who showed that higher yields were obtained in

genotypes exhibiting low NR activity. This observation is

consistent with the negative relationship between NR and

GS activity which suggests that, when the rate of nitrate

reduction is too high, GS activity becomes limiting to cope

with the stronger ¯ux of reduced nitrogen.

Understanding genetics and physiology of N utilization

from the study of QTLs

QTLs for N-uptake and N utilization ef®ciency: QTLs

detected for agronomic traits (grain yield and its

components, kernel number and kernel weight), N-content,

and some other associated traits as senescence, are recalled

in Fig. 1. It appears clearly, as shown by Bertin and Gallais

(2001) that more QTLs for yield and its components are

detected for plants grown under high N-input. By contrast,more QTLs are detected for N-content for plants grown

under low N-input. For whole-plant N-uptake at high N-

input, ®ve QTLs were detected, three on chromosome 1

and two on chromosome 4, whereas only one QTL was

detected at low N-input. This is due to a very low genetic

variation in such a condition (Bertin and Gallais, 2000)resulting from a dilution effect (high negative relationship

between dry-matter yield and N-content). For grain N-

uptake, the same situation was observed, since no QTL

were detected at low N-input. Unlike N-uptake, QTLs for

whole plant NUtE were detected mainly at low N-input.

Under this condition, the ®ve detected QTLs explained

38.9% of the phenotypic variance, whereas at high N-

input, only one speci®c QTL was detected. The same

situation was observed with QTLs detected for grain

NUtE. Four QTLs were detected under low N-input, three

coinciding with those detected for whole-plant NUtE and

two under high N-input (one coinciding with one of the

previous QTLs, the other being speci®c to plants at high N-

input). As discussed by Bertin and Gallais (2001) differ-

ences in heritabilities and effects of N-deprivation on

means are not suf®cient to explain such results. Therefore,

the assumption here is that genes are regulated differentlyaccording to the level of N-fertilization. This is also

consistent with the conclusion that genetic variability

(variances and correlations) was expressed differently

under both growth conditions.

QTLs for absorption and remobilization: At low N-input,

probably due to the high experimental error expressed by

the low heritability of such traits, only one QTL was

detected. It concerns the remobilization of nitrogen from

the stem. It is located at the top of chromosome 1 and

coincides with the gln1 gene (encoding cytosolic GS). At

high N-input, ten other QTLs were detected (Table 1). Five

are located on chromosome 1 at 80, 122, 172±182, and 204cM from its top. The ®rst three were related to N-

remobilization (expressed at either absolute or relative

values) from the whole plant at ¯owering, or from the leaf

blades or the stem. The ®rst at position 80 cM coincided

positively with a QTL of kernel weight under both levels of

N fertilization. The second, at position 122 cM coincided

negatively with a chromosome region where a QTL for

kernel number and three genes are present. Two of these

genes encodes enzymes involved in N-assimilation (NR1

and GDH1) while the other encodes ADPGppase, an

enzyme involved in C-metabolism. The two QTLs

detected in position 172±182 correspond to two types of

traits: the relative rate of N-absorption in the grain

(expressed as % of the total N-uptake in the shoots or as

% of total nitrogen in the grain) and the relative rate of

remobilization. For the absorption the favourable allele

comes from the parental line Io, while for remobilization it

origin is from the other parental line F2. However, it is

impossible to conclude whether it is two different QTLs

nearly linked or the same QTL with a pleiotropic effect

because the two traits are highly negatively correlated (r P=

±0.75**). It is interesting to note that there was coinci-

dence with the gln2 gene encoding another cytosolic GS

isoenzyme. This zone was also involved in the genetic

control of grain yield, kernel number and grain proteinyield at high N-input. The last QTL on chromosome 1 in

position 204 cM was related to absolute remobilization

from the whole plant at ¯owering and coincided with

QTLs for grain yield and kernel number at high N-input.

Another QTL located on chromosome 2 at position 64

cM was related to the N-remobilization process. It

overlapped positively with a QTL for kernel weight

detected at high N-input. On chromosome 4, three QTLs

were detected: two (at 104 and 148 cM) were related to N-




F i g .

1 .

L o c a t i o n s o f t h e d e t e c t e d Q T L s f o r a g r o n o m i c t r a i t s w i t h a p o p u l a t i o n s i z e o f 9 9 r e c o m b i n a n t i n b r e d l i n e s c r o s s

e d t o t h e t e s t e r F 2 5 2 .

A Q T L w a s s h o w n w h e n i t w a s d e t e c t e d i n o n e

y e a r o r i n t h e a v e r a g e o f t h e t w

o y e a r s .

Q T L s d e t e c t e d a t h i g h N - i n p u t a r e

o n t h e l e f t o f t h e c h r o m o s o m e w h e r e a s t h o s e d e t e c t e d a t l o w N - i n p u t a r e o n t h e r i g h t o f t h e c h r o m o s o m e . F o r

m o r e d e t a i l s o n t h e p a r a m e t e r s o f t h e s e Q T L s s e e B e r t i n a n d G a l l a i s ( 2 0 0 1 ) .

N o t e t h a t n i t r o g e n w h o l e - p l a n t c o n t e n t i s t h

e r e c i p r o c a l o f n i t r o g e n u s e e f ® c i e n c y a t t h e

w h o l e p l a n t l e v e l .




remobilization expressed either as absolute or relative

values. The ®rst one overlapped positively with a QTL of

kernel weight detected at low N-input, whereas the second

one overlaps, also positively, with a QTL of kernel weight

detected in both nitrogen fertilization conditions.

Moreover this second QTL coincided with two genes

encoding enzymes involved in nitrogen metabolism (NR2

and gln3). The last QTL on chromosome four at position

236 cM was linked to the relative rate of leaf N-

remobilization and is located near the genes encoding

PEPC and NTR1.Finally, among the ten QTLs detected for N-remobiliza-

tion, three coincided with QTLs for kernel weight and one

with a QTL for grain yield. This ®nding stresses the role of

N-remobilization during grain ®lling, despite the lack of

correlation between N-remobilization and grain yield or

kernel weight. Considering the role of post-anthesis

absorption at high N-input for grain ®lling, it is surprising

that not more QTLs were found for N-absorption. This

could mean that there are many genes involved, each

having a relatively low effect. Furthermore there were

three coincidences of QTLs for remobilization with a gene

encoding cytosolic GS which suggests that such genes are

involved in the control of remobilization. However, takinginto account the high negative correlation between post-

anthesis N-uptake and N-remobilization, a QTL for

remobilization could be considered as a QTL for N-

uptake, with an allelic effect of opposite sign. Following

results on physiological traits will shed some light on the

meaning of such QTLs.

QTLs for leaf nitrate content and NR activity: Five QTLs

for leaf nitrate content explaining 28% of the phenotypic

variation were detected: two were located on chromosome

2, both with the favourable allele from the parental line F2

and three on chromosome 5 with the favourable allele from

the parental line Io. On chromosome 2 one of the QTLs for

leaf nitrate content, coincided positively with a QTL for

kernel weight when plants were grown under high N-input.

One of the QTLs for leaf nitrate content located on

chromosome 5 was also positively coincident with a QTL

for grain yield and kernel weight, regardless of the N-

fertilization level. These results are in agreement with the

positive correlation observed between leaf nitrate contentof young developing plants, grain yield and kernel weight

in ®eld-grown mature plants independent of the level of

fertilization. Furthermore, the observed coincidences with

QTLs for kernel weight support the conclusion that nitrate

content (and quantity) at a young stage is an indicator of

post-anthesis N-uptake ability.

For maximal leaf NADH-NR activity (with measure-

ments performed only on a one-year experiment), two

main QTLs were found on chromosome 5 explaining

36.2% of the observed phenotypic variation, which is very

high for only two QTLs. One of the QTLs for NADH-NR

activity located in the region of the gln4 locus, was

negatively coincident with a QTL for nitrate content and aQTL for yield, both detected under low or high N-input.

These results are consistent both with the observed

negative correlation between grain yield or kernel weight

and leaf NR activity and the expected negative correlation

between NR activity and nitrate content. The other QTL

was positively coincident with a QTL for nitrate content.

QTLs for GS activity: Six QTLs for total leaf GS activity

were detected explaining 52.5% of the phenotypic vari-

Table 1. QTLs detected for N-remobilization and post-anthesis N-uptake with a population size of 99 recombinant inbred lines

crossed to the tester F252

Traith Chroa Positionb Markerc Intervald LODe R2 f Allele effectg

Stem remobilization 94N± 1 8 UMC11 2±24 1.91 9.7 +Leaf blade remobilization 94N+ 1 80 SC351 56±94 2.30 10.2 ±% Remobilization from leaf 94N+ 1 80 SC351 60±94 2.95 13 ±Total remobilization (94+95)N+ 1 122 SC60B 110±138 2.27 10.1 ±

% Remobilization from leaf 94N+ 1 168 SC282A 154±188 2.51 11.1 ±Total remobilization (94+95)N+ 1 172 SC282A 160±198 3.46 15 ±% Absorption in grain (94+95)N+ 1 182 SC296 160±198 2.45 10.9 +Total remobilization (94+95)N+ 1 204 ADH1 198±210 3.28 14.4 ±% Remobilization from leaf 94N+ 2 58 SC108 30±72 2.10 9.3 ±Total remobilization (94+95)N+ 2 68 SC136 60±74 3.78 16.4 ±Remobilization from stem 94N+ 4 104 SC59C 94±112 2.67 11.8 ±Remobilization from stem (94+95)N+ 4 148 UMC66 116±168 2.03 9.2 ±% Remobilization from leaf 94N+ 4 236 UMC133 202±244 2.00 13.8 +% Remobilization from stem 94N+ 6 228 MDH2 204±236 1.94 10.3 ±

a Chromosome number.b Distance in cM from the chromosome top of the maximum LOD.c Nearest marker on the genetic map (Causse et al., 1996) from the chromosome top.d LOD-1 interval.e Maximum LOD greater than 2 except for the ®rst and last line of the table. f Percentage of phenotypic variance explained by the variation at the QTL.g + when the favourable allele derives from the Io parent whereas ± when it derives from F2 parent.h 94 means observation in the year 1994, whereas 94+95 means average of the two years of study 1994 and 1995. N+ (N±) refers to high (low) N-input.




ation: three were located on chromosome 1, two on

chromosome 5, and the other on chromosome 9.

Interestingly, out of these six QTLs, three coincided with

genes encoding cytosolic GS quoted as gln1, gln2 and

gln4. This result suggests that for these three genes the

®nal leaf cytosolic enzyme activity is mostly regulated at

the transcriptional level. By contrast, for the other

cytosolic GS gene gln3 located on chromosome 4 andthe gene encoding plastidic GS (gln5) located on chromo-

some 10, other regulatory mechanisms acting at the post-

transcriptional and/or translational levels are likely to be

involved in controlling the corresponding enzyme activity

(Cren and Hirel, 1999). The detection of four QTLs for leaf

GS activity which did not coincide with GS structural

genes indicates that some loci located on different

chromosome segments may be partly involved in the

regulation of both cytosolic and plastidic GS activity.

Two QTLs for GS activity were coincident with QTLs

for yield and its components (kernel weight and kernel

number) and a GS gene. One was located on chromosome

1 coincident with gln2 locus and with a QTL for yield andkernel number at high N-input, and one on chromosome 5

coincident with gln4 locus and with QTLs for yield and

kernel weight whatever N-input. Such positive coinci-

dences are consistent with the positive correlation

observed between grain yield and GS activity, particularly

at low N-input. However, QTLs for yield on chromosome

5 can be considered as common to both nitrogen levels,

because the favourable allele was detected at both low and

high N-inputs. By contrast, the QTL for yield, coinciding

with QTL for leaf GS activity on chromosome 1, was

detected only under high N level. This could mean that

gln2 and gln4 translation products have a different role. In

the same manner, the QTL for N-remobilization in the

region of the gln1 locus (on chromosome 1), which

coincides positively with a QTL of GS activity, could be

considered as a QTL of true remobilization, whereas the

QTL for N-remobilization in the region of the gln2 locus,

which coincides negatively with a QTL of GS activity,

could be considered as a QTL of post-anthesis N-uptake.

The gln1 locus could code for an isoenzyme involved in N-

remobilization whereas the gln2 locus could code for an

isoenzyme involved in nitrogen assimilation. This supports

the conclusion that the different GS genes appear to have

non-overlapping functions in different organs or tissues

and according to the plant developmental stage(Sakakibara et al., 1992b; Li et al., 1993; Rastogi et al.,

1998). The relative contribution of the corresponding GS

isoenzyme activity in either synthesizing or recycling

organic nitrogen necessary for grain ®lling would be ®nely

balanced, not only depending on the plant developmental

stage but also on soil N availability.

The coincidence between the gln4 locus and several

QTLs for grain yield, kernel weight, leaf GS activity, NR

activity, and leaf nitrate content (Fig. 2) suggests that both

nitrate availability and the reactions catalysed by NR and

GS are key steps in the NUE for seed production.

However, the coincidence is negative between the QTL

for NR activity and all the others co-localizing with it,

whereas it is positive between them. This suggests that

complex interactions between GS and NR activities are

likely to occur. As already discussed in the previous

section, this result is consistent with the negative impact of nitrate reduction capacity on yield and its components. By

contrast, the positive coincidence between QTLs for grain

yield, kernel weight, leaf nitrate content, and leaf GS

activity, con®rm the positive effect of the last two traits on

yield found in the correlation studies. Furthermore, the

coincidence between QTLs for leaf GS activity, leaf NR

activity and leaf nitrate content found in two regions on

chromosome 5, is in favour of the hypothesis that signals

derived from the ammonia assimilatory pathway interact

with nitrate uptake and reduction (Scheible et al., 1997).

Finally gln4 appears as a good candidate gene controlling

NUE and in¯uencing yield.

It can also be underlined that, on chromosome 10, therewas coincidence between another GS locus (gln5) and

QTLs for ASI, leaf senescence, and NNI. Interestingly, this

locus corresponds to the plastidic GS. Therefore, taking

into account coincidences shown previously between QTL

for remobilization and the different members of the GS

multigene family, it appears that coincidences observed

with the ®ve GS loci are quite consistent with their possible

role. It is well known that the different genes encoding GS

can be differentially expressed according to both the

physiological status and the developmental stage of the

plant (Cren and Hirel, 1999).

QTLs for GDH : Three QTLs for GDH activity were

detected. Two corresponding to GDH aminating activity

were located on chromosome 3 and 8 while the other

corresponding to GDH deaminating activity was located

on chromosome 6. The QTL for GDH deaminating activity

was found in plants grown in the ®eld under a high level of

N-fertilization whereas the two QTLs for GDH aminating

activity were found in plants grown under low N-input.

The two QTLs for GDH aminating activity coincided

positively with QTLs for grain yield and kernel number

whereas the QTL for GDH deaminating activity coincided

negatively with a QTL for kernel number. This result

suggests that the GDH aminating activity may be animportant factor controlling plant productivity as already

found using transgenic plants overexpressing the enzyme

(Ameziane et al., 2000). GDH deaminating activity could

be involved in controlling the translocation of assimilates

during the remobilization phase, when it is induced, as

suggested by the negative correlation between GDH

activity and leaf N-content at maturity. This possible role

of GDH is strengthened by the recent ®nding that GDH

protein is mostly concentrated in the vascular tissue of a




F i g .

2 .

L o c a t i o n s o f t h e d e t e c t e

d Q T L s f o r p h y s i o l o g i c a l t r a i t s ( l e a f n i t r a t e

c o n t e n t , l e a f G S a n d N R a c t i v i t i e s o f y o u n g

p l a n t s a t h i g h N - i n p u t ; G D H a n d G S a c t i v i t i e s o f a d u l t p l a n t s a t b o t h

h i g h a n d l o w N - i n p u t ) a n d t h e i r c o i n c i d e n c e s w i t h m a p p e d g e n e s a n d Q T L s f o r g r a i n y i e l d ,

k e r n e l n u m b e r ,

t h o u s a n d

k e r n e l w e i g h t ( T K W ) , r e m o b i l i z a t i o n , a n d

p o s t - a n t h e s i s N - u p t a k e a t

b o t h h i g h ( N + ) a n d l o w - i n p u t ( N ± ) . Q T L s f o r r e m o b i l i z a t i o n a n d p o s t - a n t h e s i s N - u p t a k e w e r e d e t e c t e d o n l y a t h i g h N - i n p u t , e x c e p t t h e o n e a t t h e t o p o f c h r o m o s

o m e 1 d e t e c t e d a t l o w N -

i n p u t . T h e g e n e t i c m a p w a s p u b l i s h e d i n d e t a i l b y C a u s s e e t

a l .

( 1 9 9 6 ) . T h e p l u s a n d m i n u s s i g n s b e l o w o r a b o v e t h e s e g m e n t r e p r e s e n t i n g t h e Q T L l o c a t i o n s h

o w s f r o m w h a t p a r e n t t h e

f a v o u r a b l e a l l e l e c o m e s : p l u s f r o m I o , m i n u s f r o m F 2 .

F o r m o r e d e t a i l s o n Q T L s f o r n i t r a t e c o n t e n t a n d G S a c t i v i t y , s e e

H i r e l e t a l .

( 2 0 0 1 ) .




number of higher plants including maize (Becker et al.,

2000).

Conclusion

Bertin and Gallais (2000) concluded from both the study of

genetic correlations among traits and the detection of

QTLs for various agronomic traits that genetic variabilitywas differently expressed under high and low N-inputs.

Further physiological studies associated with the detection

of QTL con®rm such a conclusion. It appears that nitrogen

stress may allow the expression of variability controlled by

speci®c genes involved in N-remobilization, unlike high

N-input which would favour the expression of variability

controlled by speci®c genes involved in post-anthesis N-

uptake.

One of the major breakthroughs from these studies,

concerns the role of the gene encoding cytosolic GS (gln4

locus) located on chromosome 5 as a candidate gene for

which the corresponding enzyme activity in¯uences grain

®lling. Now, experiments are in progress to overexpressthe gene and to transfer the favourable allele in a genotype

with the unfavourable allele in order to verify whether

grain ®lling and grain yield is improved. Other candidates

genes among genes encoding enzymes involved in N±

metabolism are the two GS genes (gln1 and gln2) on

chromosome 1 and the GS gene on chromosome 4 (gln3).

The corresponding enzyme activity of all these three genes

could be involved either in N-remobilization or in N-

translocation during the process of grain ®lling. It remains,

however, to determine whether each metabolic process

involved either in the assimilation or in remobilization of

nitrogen is controlled by a single GS gene or a combination

of genes which can give rise to an homo- or an hetero-

octameric form of the enzyme (Hirel and Lea, 2001).

In conclusion, these results clearly show that genetic and

physiological bases of NUE can be studied in a integrated

manner by means of a quantitative genetic approach using

molecular markers, genomics, and combining both agro-

nomic and physiological studies. Such an approach leads

to the identi®cation of candidate genes to validate other

approaches such as gene transfer or mutagenesis.

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An Approach to the Genetics of NUE in Maize

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